Drosophila buzzatii and D. koepferae. Genital morphology in interspecific hybrids was examined and compared to the corresponding parental lines.The rapid evolution of genital morphology is a fascinating feature that accompanies many speciation events. However, the underlying patterns and explanatory processes remain to be settled. In this work we investigate the patterns of intraspecific variation and interspecific divergence in male genitalic morphology (size and shape) in the cactophilic sibling species D. buzzatii and D. koepferae showed contrasting patterns of genital morphological variation. Though genitalic size and shape variation have a significant genetic component in both species, shape varied across host cacti only in D. buzzatii. Such plastic expression of genital shape is the first evidence of the effect of rearing substrate on genitalic morphology in Drosophila. Hybrid genital morphology was not intermediate between parental species and the morphological resemblance to parental strains was cross-dependent.Despite of being siblings, Our results suggest the evolution of different developmental networks after interspecific divergence and the existence of a complex genetic architecture, involving genetic factors with major effects affecting genital morphology. The evolutionary processes governing the divergence of animal genitalia are mostly unknown and constitute one of the most intriguing pieces of a mayor puzzle that is speciation -5. In maThree main hypotheses have been proposed to explain the evolution of genital morphology: the lock and key, the pleiotropy and the sexual selection hypotheses. The lock and key hypothesis  states tThe pleiotropy hypothesis assumes that genital variation is largely neutral. Since genital and non-genital morphological traits are implicitly genetically correlated, changes of allele frequencies at loci pleiotropically affecting general morphology and genitalia may lead to rapid and arbitrary evolution of genitalic traits ,10,11. TDrosophila and their hybrids [The study of the evolution of male genitalia may be even more complicated not only because it may be influenced by both natural and sexual selection, but also because it's phenotypic expression might be influenced by environmental factors  as occur hybrids . TherefoDrosophila repleta group [D. buzzatii and D. koepferae [D. buzzatii is mainly adapted to breed on decaying tunas (genus Opuntia), while D. koepferae prefers the necrotic stems of columnar cacti of the genera Cereus and Trichocereus [D. buzzatii males can inseminate D. koepferae females and female hybrid offspring can be successfully backcrossed to D. buzzatii males [The aedeagus, which is the intromittent organ of male genitalia , is consta group . To thisoepferae ,20, whicoepferae , sexual oepferae . Both spoepferae ,24, howehocereus . Though ii males . Furtherii males ,27.Drosophila [The knowledge of the ecology of these cactophilic osophila ,24,28,29In this work we investigate the sources of phenotypic variation, genetic and environmental, by examining genitalic size and shape in flies of several isofemale lines of both species and interspecific hybrids raised in two different species of host cacti.D. buzzatii, 294 D. koepferae and 60 interspecific hybrids).A total of 606 males were analyzed in this study (252 D. koepferae and 12 in D buzzatii (results not shown). The cumulative contribution of the first 5 principal components of the elliptic Fourier descriptors (EFDs) of the genital outlines accounted for over 74% and 77% of total shape variance in D. koepferae and D. buzzatii, respectively and nearly 84% of shape variation in the interspecific analysis  while in D. koepferae differences between flies grown in different cactus media were not significant . As visually observed, D. buzzatii and D. koepferae significantly differed in their genitalic shape and presented morphological variation not only among lines within species but also interacting with the breeding substrate (Table We detected significant differences in aedeagus size between species but more notably between flies reared in different cacti Table . Howeverte Table .D. buzzatii the Cactus by Line interaction was significant and accounted for a relatively high percentage (12.1%) of phenotypic variance in aedeagus size. However, only the Line factor was significant in D. koepferae, neither the Cactus effect nor the Cactus by Line interaction were significant.The results of intraspecific ANOVAs also revealed important differences Table . In D. bD. buzzatii. In D. koepferae, in contrast, our results show that variation in aedeagus size has a genetic component, devoid of any plastic response in relation to the breeding substrate. According to the results of the MANOVAs, variability among lines in aedeagus shape was significant in both species , whereas in D. buzzatii, we detected a significant allometric relationship . Furthermore, aedeagus size and wing length varied isometrically in D. buzzatii as suggested by a coefficient of allometry not significantly different from 1 .We also studied the relationship between variables describing size of male genitalia and wing length. These variables were not significantly correlated in D. buzzatii and D. koepferae [D. buzzatii or D. koepferae. In all cases hybrid males failed to produce offspring even though copulation attempts were observed in the vials. Hybrid progeny obtained in crosses 4853 and 8832 could only be tested in vials prepared with the medium prepared with fermenting Opuntia due to low numbers of hybrid larvae, while in the other crosses the yield of hybrid progeny was high enough to be reared in both cactus media.Four interspecific crosses, out of 25 attempted,  yielded enough hybrid progeny as to perform the present study. These results are in agreement with previous studies reporting strong premating isolation between oepferae . In ordeOpuntia, the rearing substrate where all crosses were able to be tested. F1 hybrid males from crosses 4853 and 8832 reared in Opuntia vials presented intermediate values that differed significantly from both parental strains . In the other crosses, in which hybrids could be reared in both cacti, a significant Genotype by Cactus interaction  was only detected in cross 4855. Hybrids male progeny in this cross presented larger aedeagi than males of the parental D. buzzatii line in Opuntia, while differences between hybrids and the D. buzzatii parent were not significant in Trichocereus. In all cases, D. koepferae presented the largest genitalia in both cacti. In one cross (3512) mean genitalic size in hybrids was significantly lower than the male parental D. buzzatii line . Based on the correlation matrix, only PC1 scores were correlated with organ size in hybrids accounting for 50.4% of shape variation  were significant in all crosses Table . In Figup < 0.001 in all cases). However, neither the Cactus factor nor the Cactus by Genotype interaction were significant in the crosses in which hybrids were raised in the two cactus media (4855 and 3512).Significant shape differences among genotypes were detected in all crosses Table . Post hoD. buzzatii lines involved in successful interspecific crosses tended to be negative and those of D. koepferae positive. However, hybrids failed to present intermediate values for both shape variables simultaneously. For instance, hybrids of cross 8832 had shape scores for both PC1 and PC2 that placed them in the morphological space closer to the D. koepferae parent (Line 88) than to D. buzzatii (Line 32). On the contrary, in cross 3512 a hybrid genital morphology was more similar to D. buzzatii for PC1 (Line12) but the mean for PC2 was more extreme than any of the parental lines. Suggestively, as explained above, hybrids in 3512 also presented smaller genitalia than both parental lines.In figure F3,110=17.59; p < 0.0001; Figure In order to evaluate the degree of resemblance of the morphology of hybrids to each parental line, we calculated the Euclidean distance to the morphological centroid of each parental strain using the shape (PCs) scores of each individual hybrid. As a rough index of morphological dissimilarity, hybrids would show equal mean distances to the centroids of both parental clouds of points if they have intermediate aedeagus morphology. Expression dominance of one genome over the other would produce phenotypes resembling more closely one parental strain or the other. Morphological dominant expression was tested with an ANOVA in which the variable was the Euclidean distance of each hybrid male to the centroid of each parental species  with Cross and Parents as fixed factors. The ANOVA revealed significant differences among crosses though it should be noted that hybrid resemblance to parental strains were not independent of the cross  development of male genitalia.The first issue raised by our study is that aedeagus size and shape vary substantially in both species, and, that a significant portion of variation is genetically determined. Moreover, the inclusion, in our experimental design, of semi-natural rearing media prepared with different cactus hosts permitted the characterization of morphological variation in terms of phenotypic plasticity in both species. In this sense, phenotypic plasticity in genital morphology was evident in ic basis . Such a punctata . In contD. buzzatii and D. koepferae. In the latter, shape and size of male genitalia, as well as aedeagus size and wing length, appeared to be largely uncoupled as suggested by the low level of within and between organ allometries. These results suggest that differences among flies in wing length are not expected to be accompanied by changes in the size of the genitalia, indicating that the factors involved in development of wings and male genitalia are largely independent in D. koepferae. In contrast, aedeagus shape and size were significantly correlated in D. buzzatii, suggesting that factors affecting size (for instance the type of cactus host) may also indirectly affect the shape of the organ.Another relevant point is that patterns of allometry within (aedeagus size and shape) and between organs (aedeagus and wing size) also differed between D. buzzatii, body size related traits (such as wing length), known to be under natural selection [In election -38, are election ,39-41. Felection -44. If tD. buzzatii [Several features of the mating system, such as female remating frequency, premating time, copulation duration, interval between successive matings, and progeny numbers, have been shown to be genetically variable in buzzatii . Howeverbuzzatii  and Colebuzzatii .D. buzzatii and D. koepferae, now, we would like to examine whether our data allow a critical evaluation of the plausibility of the three main hypotheses proposed to explain genital evolution. Though our results are not entirely conclusive in this respect, the extensive phenotypic and genetic variation in aedeagus morphology are strong evidence against the "lock and key" hypothesis, that predicts low levels of variation (both phenotypic and genetic) in genital structures. However, the correlation between aedeagus morphology and wing length, along with the condition dependence (phenotypic plasticity in relation to cactus hosts) are in agreement, at least in D. buzzatii, with predictions of the hypothesis of pleiotropy. Concerning the third hypothesis, sexual selection, we must await for the results of experiments testing the relationship, if any, between genital morphology and reproductive performance.To this point we have presented basic features of the patterns of variation of aedeagus morphology in D. buzzatii and D. koepferae. In this sense, our results seem in conflict with the single available work comparing genital morphology in hybrids and parental species [D. buzzatii or D. koepferae varied among crosses. In fact, hybrid's morphological distance to D. buzzatii and D. koepferae depended on the parental strains employed in the crosses. In none of the 4 crosses hybrid morphology was phenotypically intermediate . Another non trivial point, that may complicate our interpretation is the difference in the time of divergence between the members of the two pairs of species, since development in interspecific hybrids is a result of a balance between the effects of the degree of heterozygosity and the degree of genomic coadaptation  and the outcome of the past selection pressures on the species studied . In this context, the idea that morphology (size and shape) of an organ potentially involved in species recognition (such as aedeagus morphology), might be associated to polymorphic inversions is consistent with recent theories linking chromosomal rearrangements and reproductive isolation [However, there are certain differences between our study and Liu et al's  that areer group ) in D. spulation  and therpulation , and tesied (see ). D. simears ago , while Dears ago . Finallydentical , whereas species ,23. Our its . The advantages of the use of the isofemale line technique in quantitative evolutionary genetics have been thoroughly described in [Fifteen isofemale lines (lines hereafter) of each species, derived from collections in the locality of Suyuque , were employed in the experiments outlined below. In this area both species coexist in nearly equal proportions  preceding the number of the male parental (D. buzzatii) line (e.g. the cross between D. koepferae line 48 and D. buzzatii line 55 was designated as 4855). Batches of 30 first-instar hybrid larvae were transferred to culture vials containing one of the two 'semi-natural' media. Four replicated vials were run per every combination of cactus and cross when the number of hatched larvae allowed it. The hybrid status of the descendants was ascertained by the cytological analysis of the polytene chromosomes of progeny larvae grown in vials run in parallel.Five lines of each species were randomly chosen to generate interspecific hybrids. All possible combinations were attempted . F1 hybrids were produced by crossing 25 virgin females of a 2 anesthesia.All cultures were maintained at 25 +/- 1°C with a 12:12 light/dark photoperiod until the emergence of adults. Adult flies were simultaneously collected and sexed under light COThe aedeagus and both wings were dissected from 2 to 5 males emerged in each replicate. Aedeagi were mounted on slides and photographed with a digital camera mounted on a microscope at 400 × magnification. Wings were also mounted and ventral views of wing images were captured with a digital camera attached to a binocular microscope (25 ×) connected to a computer. In each image, we scored total wing length (WL) using TpsDig .D. buzzatii and D. koepferae that preclude the possibility of determining an adequate number of reliable homologous landmarks. However, the aedeagus is a flat quitinous organ that can be effectively described in shape and size in two dimensions when flattened under a cover slip. Consequently, we decided to employ an approach based on elliptic Fourier descriptors (EFDs) [x and y coordinates of the outline of the studied organ are fit separately as functions of arc length by Fourier analysis, so that the outline can be decomposed into a weighted sum of sine and cosine functions designated as harmonics. Outlines from digital images were used to obtain Fourier coefficients for a polynomial function of 30th degree which were computed with SHAPE v1.2 package, [As shown in Figure s (EFDs)  as a pros (EFDs) . This ispackage,  using Elpackage, ,58,59. FThe area of each aedeagus (in pixels) was calculated from the digital images and considered as an estimator of the size of the organ. We performed a normalization of the descriptors based on the first harmonic ellipse that corresponds to the first Fourier approximation to the contour information (reviewed in ). Thus, The variance-covariance matrix of the 120 (4 per harmonic) estimated EFDs coefficients was used as input in a principal components analysis. This procedure allowed us to summarize the information assessed in the coefficients and reduce the dimensionality of the variables  in a lowWe worked with two sets of PC scores. The first set of principal components were calculated from the matrix of coefficients derived form the analysis of the outlines of the genitalia of males of the parental lines employed in successful interspecific crosses and the hybrids. The second set was obtained separately for each species improving the assessment of intraspecific variation in aedeagus morphology by avoiding the noise resulting from conspicuous interspecific morphological differences in the estimation of the PCs. This set was used in the evaluation of intraspecific sources of shape variation. Preliminary analyses with this technique showed that it is repeatable and reliable in species discrimination .C) may be interpreted as phenotypic plasticity, while significant differences among isofemale lines as due to genetic differences . Finally, a significant L × C interaction may be construed as an estimation of genotype by environment interaction (GEI) or more explicitly, that the response of isofemale lines is not independent of the rearing cactus.Both interspecific and intraspecific size differences were investigated by means of ANOVAs, with Species , Cactus  and Line (nested in Species random factor) as main sources of variation. The variable was log-transformed to ensure homoscedasticity. According to our experimental design, in the ANOVAs for species, a significant cactus effect .We also evaluated the allometric relationship between aedeagus size and wing length, a trait correlated with overall body size, which in the studied species are known to be affected by the rearing substrate . Size daThe principal components scores obtained by means of the general assessment of interspecific shape variation of parental lines and F1 hybrids were used in the examination of morphological patterns of variation in the offspring of interspecific crosses. Size was analyzed by means of an ANOVA and shape with a MANOVA. In both cases the principal factor was Genotype (both parents and the hybrids) and in those crosses in which hybrid larvae could be reared in both cactus hosts, Cactus was also considered as a fixed factor.Statistica 6.0  was usedIS and EH conceived the study and read the salivary gland squashes. IS and VC carried out the experimental crosses, egg collections, larval seeding, rearing and adult collection. IS dissected the male genitalia, performed the morphological quantification, statistical analyses, and wrote the first draft of this manuscript. IS and EH wrote the final version of this manuscript. VC mounted the wings on microscope slides and provided the wing measurements. VC and JF helped to draft the final version of this manuscript. All authors read and approved the final manuscript.


Drosophila melanogaster wings in an experimental design with extensively replicated and fully controlled genotypes. The amounts of variation among individuals and of fluctuating asymmetry differ markedly among genotypes, demonstrating a clear genetic basis for size and shape variability. For wing shape, there is a high correlation between the amounts of variation among individuals and fluctuating asymmetry, which indicates a correspondence between the two types of buffering. Likewise, the multivariate patterns of shape variation among individuals and of fluctuating asymmetry show a close association. For wing size, however, the amounts of individual variation and fluctuating asymmetry are not correlated. There was a significant link between the amounts of variation between wing size and shape, more so for fluctuating asymmetry than for variation among individuals. Overall, these experiments indicate a considerable degree of shared control of individual variation and fluctuating asymmetry, although it appears to differ between traits.The nature of developmental buffering processes has been debated extensively, based on both theoretical reasoning and empirical studies. In particular, controversy has focused on the question of whether distinct processes are responsible for canalization, the buffering against environmental or genetic variation, and for developmental stability, the buffering against random variation intrinsic in developmental processes. Here, we address this question for the size and shape of  Developmental buffering is an important factor in evolutionary processes, because it can maintain adaptive phenotypic traits in the presence of genetic and environmental variation and it can conceal genetic variation from selection Empirical studies have tackled the question of whether canalization and developmental stability are distinct processes by comparing variation among individuals and the left-right asymmetries within individuals. Two main approaches have been used, which focus either on the amounts of variation or on covariance structures of multivariate features such as shape. Some studies have indicated that the amounts of individual variation and fluctuating asymmetry (FA) are correlated among genotypes Drosophila melanogasterThis study used both these approaches simultaneously in the context of an experimental design with complete control of genetic variation, replicated for 115 distinct genotypes from the Exelixis deficiency stocks of We digitized 15 landmarks on the left and right wings of each fly . To estir = 0.81, P<0.0001) and for FA . Although the two measures are computed from different aspects of variation, they both convey similar information in the context of this study and therefore can be interpreted as nearly equivalent measures of shape variation.We used two different methods to quantify the amounts of shape variation among individuals and FA F114, 259 = 2.08, P<0.0001) and for FA . Similarly, the ANOVAs for both measures of shape variation indicated significant effects of the genotypes on variation among individuals  and on FA . These results show that the chromosomal deficiencies have clear effects on the amounts of variation among individuals and on FA, which in turn indicates a genetic basis for the amounts of variation.The amounts of variation differed markedly among the different genotypes, although there was also a consistent effect of the vials in which the flies had been reared. For centroid size, the ANOVAs indicated that the variation among genotypes exceeded the variation among vials both for variation among individuals (P<0.0001), and in the analysis using Mahalanobis distance, it was 0.67 (P<0.0001). Overall, there is a clear trend for genotypes with greater amounts of individual variation to have greater amounts of shape FA as well.For both measures of shape variability, the amounts of shape variation among individuals and of shape FA were significantly correlated across genotypes . In the r = 0.074, P = 0.22; In contrast, the correlation between individual variation and FA of centroid size was low and not statistically significant (P<0.0001 in permutation tests). Accordingly, genotypes that are more asymmetric for size also tend to be more asymmetric for shape. The correlations between amounts of individual variation of size and shape were 0.25 (P = 0.0059) and 0.19 (P = 0.022) for the measures using Procrustes and Mahalanobis distances, respectively  and size accounted for an average of 8.18% of shape variation among individuals. The asymmetry of size accounted for an average of 4.61% of the asymmetry of shape . Accordingly, size accounts for only relatively minor proportions of shape variation and asymmetry under the conditions of our experiment. It therefore appears that the correlation between size and shape in the amounts asymmetry and individual variation is not simply the result from a direct allometric link between size and shape, but is based at least to a considerable part on linkages in the processes that produce or buffer against the variation.To assess the possibility that this association was caused by a direct developmental link between size and shape, we tested for allometry within genotypes by multivariate regression of shape on centroid size P ≤ 0.0001).To examine whether among-individual variation and FA primarily concern the same or different features of shape, we quantified the degree of congruence between the respective patterns of covariation in landmark shifts. For this purpose, we computed matrix correlations between the respective covariance matrices for those 95 genotypes for which there were at least 50 specimens. Matrix correlations were computed both with the diagonal blocks included and excluded to examine whether the total patterns of landmark variation differ from the patterns of covariation among different landmarks This study shows a significant genetic effect on the amounts of individual variation and FA as well as a clear association in both the amounts and patterns of variation between individual variation and FA for wing shape. This is consistent with the idea of a common genetic and developmental basis for buffering of wing patterning processes against variation from different sources. The correlations between size and shape in the amounts of FA and individual variation provide further evidence in favor of a common basis for buffering. In contrast, the lack of association between the amounts of individual variation and FA for centroid size indicates that these relationships depend on the specific traits under study and the processes involved in their development. Here we discuss these findings and their implications for interpreting the mixed results of published empirical studies on canalization and developmental stability.Drosophila melanogasterDrosophila wing found an association between the severity of effects and levels of FA Our data indicate a clear association of the amounts of individual variation and FA of wing shape across genotypes . This fiIn stark contrast to the shape data, the association between individual variation and FA did not hold in the analysis for centroid size . This diThe discrepancy between these findings for size and shape highlights a methodological problem inherent in studies of developmental buffering: how can the effects of buffering be distinguished from differences in the initial input of developmental variation? Buffering is only observable if there is variation, and the resulting phenotypic variation is the joint expression of both the input of variation and the buffering of that variation by the developmental system. The original amount of variation, however, which is the input for the buffering processes in the developmental system, is unknown. The input of variation and buffering are therefore almost inextricably linked and cannot be separated without specifically designed experiments. Here we used samples of flies with controlled genotypes, so that genetic variation within samples can be ruled out. However, non-genetic effects cannot be controlled in this manner. The theoretical limit is a situation in which the environment is held constant so that the conditions under which the wings of two different flies develop are no more different than the conditions encountered by the two wings of the same fly. In this case, the left and right wings of individual flies would not be correlated, and the variance for individuals, var(0.5(right+left)), would be one-quarter of the variance for asymmetry, var(right−left). For shape, individual variation (quantified using Procrustes distance) exceeded this theoretical limit consistently, but only by relatively small amounts , indicatThe correlation between the amounts of FA for centroid size and shape across the 115 genotypes exceeds the within-sample correlations of size and shape asymmetry for all but a few samples. Therefore, the direct developmental association of size and shape is not sufficient to account for the agreement of amounts of FA of size and shape. This is further evidence for a common genetic control of developmental variation of size and shape, although the data do not permit one to distinguish whether this control affects the origin of developmental noise or the developmental stability buffering against it. The association across many deficiency genotypes affecting different genomic regions may also be taken as evidence that a range of different genes contribute to the control of developmental stability, rather than just a few specialized genes Drosophila melanogasterDrosophila species Drosophila subobscura found considerable differences We not only compared the amounts of variation, but also the patterns of landmark shifts associated with individual variation and FA. There is a close and consistent correspondence between the patterns of individual variation and FA. A similar correspondence of patterns of shape integration for individual variation and FA has been found previously in the wings of Because each of our samples was genetically uniform, we can rule out a contribution from allelic differences to the variation among individuals, which would produce effects that depend on the genetic composition of the sample and usually would differ from the within-individual effects. Imagine a population in which one locus with two alleles affects shape, so that the allelic differences will cause variation along a single line (with additive effect only) or in a plane (with additive and dominance effect). Unless the non-genetic components of variation also happen to be concentrated in the direction of this particular line or plane, the two components of variation will therefore be different. Even when more complex genetic models are used, the covariance structure among individuals will depend on the particular mix of genotypes, and may not reflect the inherent patterns of canalization. This reasoning can explain the closer resemblance of the patterns of FA to those of environmental rather than of genetic variation that has been found in empirical studies that specifically examined this effect Overall, the results of this study clearly indicate that both the amounts and the patterns of individual variation and FA of shape are associated consistently across a broad spectrum of distinct genotypes. This suggests that canalization and developmental stability for wing shape share a common basis The flies used here were offspring from crosses between the Exelixis deficiency stocks A set of 15 landmarks was digitized on each image . To asseThe shape information was extracted from the landmark coordinates with a generalized least-squares Procrustes fit To quantify individual variation and FA of wing size, we used the within-sample variance of the centroid size We used two different methods to quantify variation, which are based on different measures of morphological distance: Procrustes distance and Mahalanobis distance To test whether the amounts of variation differed among genotypes, we used an extension of Levene's test To examine the correspondence between the amounts of individual variation and FA, we computed the variances based on the two distance measures for data sets with either the mean shapes of both wings or the signed (right−left) differences of wing shape. Product-moment correlations were then computed across genotypes. The statistical significance of correlations was assessed with permutation tests Allometry within genotypes was tested by multivariate regression of shape on centroid size x and y coordinates of each landmark were kept together) x and y coordinates of each landmark) For the strains for which at least 50 specimens were available, we also compared the patterns of shape variation between individual variation and FA Table S1Strains used in this study and various sample statistics.(0.36 MB DOC)Click here for additional data file.


In this paper we present a novel approach to quantifying genetic architecture that combines recombinant inbred lines (RIL) with line cross analysis (LCA). LCA is a method of quantifying directional genetic effects  that differentiate two parental lines. Directional genetic effects are thought to be critical components of genetic architecture for the long term response to selection and as a cause of inbreeding depression. LCA typically begins with two inbred parental lines that are crossed to produce several generations such as F1, F2, and backcrosses to each parent. When a RIL population (founded from the same P1 and P2 as was used to found the line cross population) is added to the LCA, the sampling variance of several nonadditive genetic effect estimates is greatly reduced. Specifically, estimates of directional dominance, additive x additive, and dominance x dominance epistatic effects are reduced by 92%, 94%, and 56% respectively. The RIL population can be simultaneously used for QTL identification, thus uncovering the effects of specific loci or genomic regions as elements of genetic architecture. LCA and QTL mapping with RIL provide two qualitatively different measures of genetic architecture with the potential to overcome weaknesses of each approach alone. This approach provides cross-validation of the estimates of additive and additive x additive effects, much smaller confidence intervals on dominance, additive x additive and dominance x dominance estimates, qualitatively different measures of genetic architecture, and the potential when used together to balance the weaknesses of LCA or RIL QTL analyses when used alone. Genetic architecture is a broad term for all factors that influence the determination of phenotype from genotype. It includes all genetic effects on traits: the number of genes, allelic effects, epistasis, pleiotropy, and genotype x environment interactions Studies of genetic architecture have revealed that epistasis, i.e. interactions between loci, is a common component of most quantitative traits. For example, biomedical studies have shown an epistatic genetic basis for many human diseases directional, effects of all loci contributing to a trait. Line crosses have become more popular in recent years as interest in quantifying epistasis in quantitative traits has increased; this method offers far greater statistical power than variance component analyses previously used to measure epistasis Line cross analysis (LCA) is a well established method of quantifying genetic architecture with a long history of use in agriculture. Because of its utility for gene discovery, much recent work has focused on understanding genetic architecture at the level of individual loci or QTL. LCA in contrast measures the summed, i.e. In this paper we present a novel approach to quantifying genetic architecture that combines recombinant inbred lines (RIL) with line cross analysis. When RIL are used in line cross analyses, scaling tests can be constructed for non-additive genetic effects with far more precision than traditional methods of estimation. The RIL can be simultaneously used for QTL identification. These two uses of a RIL population yield qualitatively different information about genetic architecture and can be used in a powerful and complementary manner.Line cross analyses typically begin with two inbred parental lines that are crossed to produce an F1 generation. F2s, backcrosses, and other generations can be produced as well; the directional genetic effects  to be estimated are limited by the number of generation means measured. For example, estimating the mean, additive, dominance, and 3 pairwise epistatic effects requires at least 6 generation means for estimation and 7 for hypothesis testing.S) and the hybridity index (θH), multiplied by the additive (A), dominance (D) or epistatic interaction effects  that potentially differentiate the parental lines (equation 1).S determines the coefficients of the additive effects' contribution to each generation's phenotypic mean. The source index is scaled from one to negative one and indicates the proportion of genes in the generation that came from parent one (P1), with +1 indicating 100% and −1 indicating 0%. P1's θS = +1 while for F1s, F2s, and RILs θS = 0.Line cross analyses are primarily carried out using frameworks based on the F2 model of Cockerham H = +1, while parents have θH = −1. The hybridity index determines the contribution of the dominance effects to each generation mean. The hybridity index is also scaled from +1 to −1, with +1 indicating that every locus is heterozygous and −1 indicating that every locus is homozygous. F1s thus have θTo this traditional set of line cross generation means, the mean of a RIL generation can be added. In this context, ‘RILs’ or a ‘RIL population’ is a set of genotypes of highly inbred F2 lines. If these genotypes were replicated, the means of each genotype can be used as individuals for calculating the overall RIL generation mean. RILs asymptotically approach complete homozygosity for all loci as the number of generations of inbreeding approaches infinity. In practice, the convention is to use six to eight generations of inbreeding, resulting in ∼99.84 to 99.96% homozygosity respectively. A major advantage of RILs is that the descendents of any one RIL are genetically identical, hence “immortal” (ignoring mutation accumulation), allowing RILs to be marker-genotyped once and phenotyped repeatedly in multiple labs and experiments. In the framework of LCA, RIL can be used to greatly improve power in estimating non-additive genetic effects.The F2 generation has a value of zero on both the source and hybridity indices. All genetic effects are scaled relative to this F2 generation mean, thus the linear contrasts used to estimate the genetic effects are sometimes called F2 scaling tests. The expected mean of the F2 and RIL generations are identical and their source indices are both zero -1, assuming equal spacing of markers throughout the genome. In the absence of segregation distortion, this will be very close to zero). However the F2 hybridity index has zero value, while the RIL hybridity index is in contrast approximately negative one. .Products of the source and hybridity indices determine the coefficients for interactions between additive and dominance effects (i.e. epistasis). For example, the product of the additive coefficient (source index) and the dominance coefficient (hybridity index) is the coefficient for the additive x dominance epistatic effect. The coefficients for additive, dominance and pairwise epistatic effects for 7 commonly used generation means are given in Xiz indicates the phenotypic mean of the iXth generation :Using equation (1) and the first six generations in Note the equations for D and AD in Lynch & Walsh (By incorporating the RIL generation's equation (for the contributions of the various genetic effects to the RIL generation mean) in the F2 scaling tests, we can construct tests for non-additive genetic effects with fewer terms than traditional tests, shown by contrasting equations (2) and (3). Incorporating the RIL means equalizes the number of generation means necessary to estimate the additive and dominance effects, and the number of generation means necessary to estimate the AA, and DD epistatic effects. This is important in providing equanimity in the power of tests for both intra- and interlocus additive vs. dominance effects; estimates of A and D both require two generation means while AA and DD both require three generation means. AD is the sole equation which retains four generation means in its estimator because the RIL mean cannot be used to simplify the equation.D follows a t distribution with 1 df. Similar test statistics can be constructed for each genetic effect following the same format.T-tests can be used to test the null hypothesis that a genetic effect equals zero, assuming that the test statistic is normally distributed under the null hypothesis. The test statistic is simply the estimated genetic effect estimate divided by the standard error of the estimate. For example, the test statistic for the composite dominance effect (using eq. (2)) isThe effect of reducing the number of terms becomes clear when we look at the new RIL-based test statistic for the composite dominance effect:2 * Var (A) + d2 * Var (B), provided that the terms being summed are independent. We can compare the variances associated with the traditional formulae for D, AA, and DD from equations (2) with the corresponding RIL equations (3). For these comparisons, we assume that all generation means have equal variance (i.e. σ2 = Var (P1) = Var (P2) = Var (F1) = Var (F2) = Var (B1) = Var (B2) = Var (RIL)).Recall that the variance of a sum equals the sum of the variances multiplied by the square of the coefficients, i.e. Var (cA + dB) = cBased on this assumption, the RIL-based equation for D, AA, and DD have 92%, 94%, and 56% reductions in variance respectively relative to the traditional equations . The varY), a design matrix (X) of coefficients derived from the source and hybridity indices, and a vector of composite genetic effects (β) to be estimated. Initially, β contains the mean and the composite additive effect and X contains two corresponding columns. An estimate of β is calculated using (XTX)−1XTy (or (TV−1XX)1−TV−1yX, where −1V is a diagonal matrix of squared standard errors for generation means if sample sizes are unequal). This estimate of β is premultiplied by X to produce a vector of predicted generation means Ŷ, given an additive genetic architecture. Ŷ is then compared with the observed Y using a chi-squared test. If the observed and predicted Y's are significantly different, then the additive model is rejected and an additive and dominance model is tested next. A new β vector containing the mean, the composite additive effect, and the composite dominance effect is estimated and multiplied by an X matrix with 3 columns to produce a new Ŷ. Increasingly complex models of genetic architecture are tested until the predicted and observed vector of generation means is not significantly different.Frequently, line cross experiments are analyzed using joint scaling tests e.g. . The joiTo illustrate the advantages of using RIL in a joint scaling context, we used seven generation means to estimate a model of additive, dominance and pairwise epistatic effects:Y = Xβ, whereβ = (XTX)−1XTy. When we used Mathematica β in terms of the generation means, the solution is:The general formula for solving linear equations is As in the individual scaling tests, the variance of the dominance effect and the additive x additive effect in RIL models are reduced by 92% and 94% respectively relative to the traditional equations. When the genetic effects are estimated simultaneously using RILs in the model above, the variance of DD is now reduced by 79%  and the variance of the estimate of the mean is reduced by 63%.More precise estimation of non-additive genetic effects will help distinguish whether these non-additive effects are rarely detected within micro-evolutionary studies because they are uncommon or because experimental designs have lacked sufficient statistical power to detect them.Phylogenetically broad crosses have gained increasing importance in both plant breeding and evolutionary genetic studies e.g. . DirectiWe show in this paper that the inclusion of a RIL generation in line cross analysis can greatly increase the accuracy with which D, AA, and DD interactions are estimated. The accurate estimation of gene interaction effects can be of substantial value for those interested in describing genetic architecture and its role in a variety of evolutionary processes Arabidopsis thaliana C24 and Col-0 genotypes to produce F7 recombinant inbred lines, then crossed these RIL to both parents and F1 in what is known as a triple test cross (TTC) design. RIL, RIL X C24, RIL X Col-0, and RIL X F1 generations were all used in line cross analysis and their results suggested that pairwise and higher order epistasis are important components of the genetic architecture of heterosis for biomass in C24 X Col-0 Arabidopsis lines. While the TTC design allows one to estimate non-additive genetic variance components, these additional crosses are not necessary to reap benefits of using RIL in LCA. We suggest purchasing RIL from stock centers to reduce the time consuming crosses necessary for more complex breeding designs.A reviewer has pointed out that one research group has previously incorporated RIL into line cross analysis. Kusterer et al. The reductions in variance used as an illustration in this paper are predicated on the assumption of equal variances in the estimate of every generation line mean. This is not necessarily a realistic assumption, particularly for the RIL generation. First, RIL populations are perforce large. The best RIL populations in many species contain 200–400 RILs and these are often grown and measured in multiple replicates for the purpose of QTL analysis. Line cross generation means are typically calculated with far fewer measures and hence degrees of freedom. Thus we might expect the variance of the mean to be substantially smaller for the RIL mean than for other generations. However, RIL populations very often show transgressive segregation, even when the parents are phenotypically similar. In fact Rieseberg et al. If a RIL population is used within a line cross analysis, little extra work is required for QTL mapping. The QTL mapping results will give qualitatively different information on genetic architecture, information that compliments the results of the line cross analysis. QTL mapping can potentially find the number of regions with additive effects (QTL) and the magnitude of those effects, as well as additive x additive epistatic regions responsible for the composite effects detected in line cross analysis. Additionally, QTL mapping may detect loci with equal and opposite effect that are invisible to LCA. For example, if the P1 allele at locus A adds 5 units to the phenotype but the P1 allele at locus B reduces the phenotype by 5 units, LCA will not detect this zero net additive difference between parents. Such canceling effects are clearly often present, evidenced by RIL population parents having very similar phenotypes but widely transgressive segregation in the inbred F2 descendents  of opposite sign invisible to line cross analysis. It can also detect additive and additive by additive epistatic QTL. It can be used to find the location of QTL for effects detected in line cross analysis. Recombinant inbred lines can be purchased from stock centers so that the time and work required to produce them is avoided. QTL studies that wish to incorporate additional line crosses will only require small increase in sample size on the order of 20%. On the other hand, line cross studies will require adding a much larger sample size to add a set of RIL lines large enough for QTL mapping. But these additional organisms phenotyped will not require the time-consuming crosses. Adding line crosses to a QTL experiment or a RIL population to a line cross experiment results in a large increase in ability to measure genetic architecture that will more than justify the modest increase in research effort and cost. Increased statistical power, qualitatively different measures, cross-validation of results, and potential to overcome weaknesses of each approach alone makes this a very powerful approach to gaining a fuller understanding of genetic architecture.


The accuracy by which phenotype can be reproduced by genotype potentially is important in determining the stability, environmental sensitivity, and evolvability of morphology and other phenotypic traits. Because two sides of an individual represent independent development of the phenotype under identical genetic and environmental conditions, average body asymmetry (or "fluctuating asymmetry") can estimate the developmental instability of the population. The component of developmental instability not explained by intrapopulational differences in gene or environment (or their interaction) can be further defined as internal developmental noise. Surprisingly, developmental noise remains largely unexplored despite its potential influence on our interpretations of developmental stability, canalization, and evolvability. Proponents of fluctuating asymmetry as a bioindicator of environmental or genetic stress, often make the assumption that developmental noise is minimal and, therefore, that phenotype can respond sensitively to the environment. However, biologists still have not measured whether developmental noise actually comprises a significant fraction of the overall environmental response of fluctuating asymmetry observed within a population.Aphis gossipyii, it was discovered that fluctuating asymmetry in the aphid wing was nearly four times higher than in other insect species. Also, developmental noise comprised a surprisingly large fraction (≈ 50%) of the overall response of fluctuating asymmetry to a controlled graded temperature environment. Fluctuating asymmetry also correlated negatively with temperature, indicating that environmentally-stimulated changes in developmental instability are mediated mostly by changes in the development time of individuals.In a morphometric study designed to partition developmental noise from fluctuating asymmetry in the wing morphology of a monoclonal culture of cotton aphid, The amount of developmental noise revealed in this trait potentially does interfere with a substantial amount of the sensitivity of fluctuating asymmetry to change in temperature. Assuming that some genetic-based variation in individual buffering of developmental instability exists in natural aphid populations, the amount of internal developmental noise determined in this study could also substantially reduce evolvability of the aphid wing. The overall findings here suggest that individual response to the seemingly high cost of stabilizing some aspects of the phenotype may account for the frequent observation of trait and species specificity in levels of fluctuating asymmetry. Phenotype is determined partly by the interaction of genotype and environment and partly by random internal noise during development. The phenotype is also generally robust to the combined effects of mutation, environmental change, and internal noise . This roDevelopmental instability most often is estimated by fluctuating asymmetry (FA), the right and left side difference in size or shape in a single trait across the population ,11-13. GBiologists have often assumed either that FA is driven mostly by uniform individual responses to a variety of stessors encountered by a natural population in a heterogeneous environment or that FA is driven mostly by variability among individual capacities to buffer against a relatively uniform level of stress presented by a homogenous environment. This difference of opinion as to whether FA is mostly environmentally-based or has a significant genetic component is debated ,15, and Drosophila imaginal wing disc over an average distance of only 2–10 cells (2–4 doublings) regardless of the stage of development. This large difference between adult body size and the comparatively small extent of synchronized cell behavior implies a large potential for the accumulation of errors caused by random differences in the timing of cell cycles within expanding cell populations during growth and development. This source of developmental noise is amplified strongly by expanding populations of growing cells during the exponential growth phase, causing the multiplicative accumulation of developmental error. Without mechanisms to regulate this kind of noise, the ascertainment of an accurate and symmetric phenotype would be nearly impossible. However, empirical observations of the symmetry of various morphologies demonstrates that most organisms actually exhibit low levels of developmental instability, as estimated by FA. Clearly, while the potential for error that leads to developmental noise must be regulated dynamically during the growth process . Milan et al.  has demoess (see ), it is Despite the likely contribution of multiplicative error to FA, it has traditionally been assumed, that developmental instability somehow originates at a subcellular molecular level and that these effects are independent and additive in their contribution to overall body asymmetry [,12 and  but alsohe "law" . Hence, he "law"  rationalhe "law" ,29. Babbhe "law"  has alsoDespite the potential for individual differences in gene and environment to influence the developmental stability of the phenotype, almost nothing is known about the overall level of internal developmental noise in a typical phenotypic trait. More importantly, how does this level of noise compare to the response of fluctuating asymmetry observed when an organism's environment changes? This is the central question in this investigation, which reports the percentage of variation in FA due to noise compared to the environmental response of FA along a temperature gradient in a genetically homogeneous population of organisms with complex morphology.Aphis gossipyii, is characterized. This species can reproduce parthenogenetically (apomictic) and often produces wings that are easily measured using multiple landmarks. Cotton aphids demonstrate large visible variation in body size, wing size, and wing FA, even within monoclonal cultures and are one of the few insects that demonstrate quite visible wing asymmetry within many individuals. Cotton aphids are also phenotypically plastic in response to temperature, producing smaller lighter morphs at high temperatures and larger, darker morphs at low temperatures. This unique feature allows observation of two genetically homogeneous groups in which differences in gene expression exist (causing the two color/size morphs). Therefore, this model system can allow for partitioning of the effect of variation in gene expression from variation in genotype on developmental stability.In this study, both the noise component of FA and its response to environment (temperature response) in the cotton aphid, Aphis gossipyii Glover) was obtained from Dr. J.P. Michaud in Lake Alfred, Florida and was brought to the Department of Entomology and Nematology at the University of Florida. The culture was maintained on cotton seedlings (Gossipium) grown at different temperatures  under artificial grow lights (14L:10D cycle). Because of potential under-sampling caused by a non-normal distribution of FA , a secoDevelopment time for individual apterous cotton aphids (Lake Alfred clone) were determined on excised cotton leaf discs using the method of Kersting et al. . Twenty In each temperature treatment, single clonal populations were allowed to increase on plants until crowded, in order to stimulate alate  production. Temperature treatments above 17°C produced small light colored and tended to feed on the undersides of leaves of cotton seedlings. Temperature treatments below 17°C produced larger, dark morphs that tended to feed on the stems of cotton seedlings. Alatae were collected using small brushes dipped in alcohol and were stored in 80% ethanol. Wings were dissected using fine insect mounting pins and dry mounted as pairs on microscope slides. Dr. Susan Halbert at the State of Florida Department of Plant Industry in Gainesville, FL performed species identification.Specimens were dried in 85% ethanol, and pairs of wings were dissected (in ethanol) and air-dried to the glass slides while ethanol evaporated. Permount was used to attach cover slips. This technique prevented wings from floating up during mounting, which might slightly distort the landmark configuration. Dry mounts were digitally photographed. Six landmarks were identified as the two wing vein intersections and four termination points for the third subcostal.Wing vein intersections were digitized using TPSDIG version 1.31 . SpecimeFA1, FA2 and FA3) of the total sample. All subsequent statistical analyses were performed using SPSS Base 8.0 statistical software [in a smaller subset , and "response" refers to the overall variance of FA between temperature treatments . The fraction of FA due to noise was calculated as the intra-individual variance of FA within each treatment divided by the total variance of FA among all individuals in the study.The overall fraction of FA due to developmental noise was 49.7% and 50.1% for size and shape FA, respectively, and the overall noise-to-response ratio was 1.00 and 1.02 for size and shape FA, respectively. There were no significant differences in the levels of developmental noise between light and dark morphs regardless of whether FA was measured using multivariate size or shape , and mean isogenic FA (both size and shape) was highly significantly different across temperatures  in the Gainesville FL clone  but this difference is due mostly to elevated FA in the 12.5°C group  morphs. Temperature trends in mean FA, which are sloped differently in the two color/size morphs Figure , were alBoth environmental temperature and FA potentially interact with growth rate and size in ectothermic species and, therefore, they should indirectly influence each other. There are several ways that FA might respond to temperature. First, increased temperature may increase molecular perturbation, which may further act to increase overall levels of developmental noise during development. This should predictably increase FA with temperature. Second, increased temperature may act as a behavioral cue to shorten development time , therebyOnly a few studies have directly investigated the relationship between FA and temperature. The results are conflicting. FA is found to increase on both sides of an "optimal" temperature -44, to bIn this study, there is a clear overall decrease in FA with increasing temperature. This strongly supports that developmental instability is a function of developmental time rather than growth rate. Thus, the longer time spent in development and therefore, the more interrupted the growth process becomes, the larger the accumulation of multiplicative developmental errors. This was especially evident in the curve of centroid size-based FA, which closely follows the curve of development time. The slope of the temperature trend in mean FA is much steeper in dark morphotypes, which also have significantly higher FA, further indicating that environmental influences on FA are primarily related to individual developmental time. This result is consistent with the explanation of the basis of FA by Emlen et al.  and counThis research indicates that within at least one morphological trait, the aphid wing, moderate levels of internal stochastic developmental noise do exist and potentially could impart some degree of insensitivity of FA to the environment. It perhaps also suggests that developmental accuracy may come at some significant cost that some organisms are unwilling to allocate towards certain aspects of their morphologies. Variation imposed by differences in this cost may account for the trait and species specificity of FA often observed among studies. Recently, it has been demonstrated that stochastic events occurring during the rapid growth phase of development can have profound effects on statistical qualities of fluctuating asymmetry, such as tail size and variance . This al


In this essay, Abigail Shearin and Elaine Ostrander discuss the proposed genomic mechanisms for the extraordinary level of phenotypic variation observed in the domestic dog and the evidence detailing the variants responsible for the many shapes, sizes, textures, and colors of man's best friend. As a result of domestication, selection for desirable phenotypes, and breed propagation, the domestic dog is unmatched in its diversity as a land mammal. Exhibiting extraordinary levels of both interbreed heterogeneity and intrabreed homogeneity, evidenced in part by the extensive linkage disequilibrium observed in many breeds, the dog provides an as-yet unrealized opportunity to uncover the molecular mechanisms that govern natural variation across mammalian species. We herein discuss recent advances in canine genomics that have made exploration of genetic mechanisms controlling breed-specific differences possible. We consider some examples where molecular mechanisms controlling simple traits have been uncovered. Finally, we reveal how combinations of genes produce complex phenotypes that can be revealed through studies of dog breeds featuring specific traits.As Darwin himself noted, the domestic dog displays a remarkable level of phenotypic diversity Canis familiaris, and possess a 2.8 Gb genome featuring 38 autosomes and the sex chromosomes, similar in size to the 3 Gb human genome. Dogs of any breed can, for the most part, be crossed to produce fertile offspring. Breeds were developed largely during the Victorian era, with special selection for both morphologic traits based on size, proportion, coat, etc., as well as behavior. To be a registered member of a breed, both of a dog's parents have to be registered members of the same breed, and their parents in turn must be registered members of the breed. Thus, each breed is effectively a closed breeding population that offers many statistical advantages for doing genetics beyond what can be done in studies of human populations There are over 300 dog breeds identified worldwide, with nearly 170 recognized in the United States by the American Kennel Club (AKC) In this essay we consider some of the features of the canine genome relevant for successful studies of selected traits. We discuss current hypotheses regarding the development and maintenance of genetic variation in dogs today. We consider examples in which identified genes account for unique, and sometimes complex, phenotypes. Finally, we consider the implications of these findings for studies of true complex traits, such as those associated with behavioral genetics.−4, which is essentially the same high level of nucleotide diversity reported in the human population. As expected, however, the level of genetic diversity within any single breed is considerably less than the species as a whole The canine genome was sequenced to both 2× The loss of diversity reported by Gray et al. The extensive LD that characterizes dogs means that genome-wide association studies (GWAS) can be done in the dog with as few as 20,000–30,000 single nucleotide polymorphisms (SNPs), compared to the million needed for the more outbred human population To aid in the selection of breeds for any given study we recently did a cluster analysis of 132 dog breeds and showed that breeds divide into five major groups: Asian and ancient dogs; hunting and gun; mastiff and terrier; herding and sight hound; and a mountain group The closest relative to the domestic dog is undeniably the gray wolf, from which the dog differs by only 0.04% in nuclear coding-DNA sequence Alx-4 gene, which is postulated to be responsible for their characteristic rear digit polydactyly Alx-4 allele, while one Great Pyrenees who lacked rear digit polydactyly did not carry the variant allele The degree to which new mutations have played a role in the development of the modern dog still requires intense scrutiny, but three major sources of genomic variation have been proposed as contributors to the high levels of phenotypic variation observed in today's domestic dogs. The first is variability associated with microsatellites or simple sequence repeats (SSRs). Fondon and Gardner hypothesized that repeat length polymorphisms, particularly those occurring in regulatory regions, were an important source of morphologic variation, in part because they occur at a mutation rate 100,000× greater than SNPs Elaborating on this theme, the same investigators found that members of the Canidae family possessed elevated genome-wide basal slippage rates, the rate at which DNA replication machinery creates new alleles due to errors in replicating repeat elements, compared to humans, non-human primates, and other members of the Carnivora order  Another mechanism that clearly accounts for a subset of diversity between breeds is carnivore-specific short-interspersed nuclear elements (SINEs) fibroblast growth factor-4 (fgf4) retrogene, a gene copied by reverse transcriptase from processed mRNA and inserted into the genome, which we demonstrated is associated with chondrodysplastic breeds displaying disproportionately short limbs FGF4 but none of the introns or regulatory machinery. Expression studies revealed that the adjacent genes were expressed in neonatal chondrocytes, as was the retrogene. However, the retrogene was not expressed in the cartilage of mature dogs. Although expressed retrogenes are common in insects, this is the first example we are aware of in which alleles of a retrogene segregate in a mammalian species such that they are a major source of morphological variation Other possible mechanisms of variation in the dog are common to many species and include mutational hotspots, chromosomal fission, and gene duplications. The latter are particularly interesting. For instance, duplication of a 133-Kb region spanning three fibroblast growth factor genes was shown to be associated with the appearance of a characteristic ridge on the back of the Rhodesian ridgeback breed The first large-scale genetic studies of canine skeletal morphology were done by Chase et al., in the Portuguese Water Dog (PWD) insulin like growth factor-1 (IGF-1) gene IGF-1 haplotype was shared among all small dogs, suggesting that a single ancestral mutation had been selected for in the development of all small dog breeds studied. Two distinct IGF-1 haplotypes segregated in large dogs, suggesting a more complicated scenario for enlarging breed size We showed that the primary signal for body size (PC1) was a four million base pair (bp) locus spanning several genes on CFA15 cis-acting mechanisms, although the critical mutation remains to be found.Additional skeletal traits studied include PC2, which defines leg length versus width in the original study of Chase et al., R-spondin-2 (RSPO2) gene is strongly associated with wire hair and “furnishings”, the latter being the moustache and eyebrows characteristically seen, for instance, in the schnauzer fibroblast growth factor-5 (FGF5) gene. Curly versus straight fur is associated with a coding SNP within the keratin71 (KRT71) gene Some of the most exciting progress in understanding the genetics of variation in dogs relates to the complex traits of coat texture and color. We recently showed that variation in canine pelage, including pattern, length, curl, and texture (smooth versus wire), are controlled by combinations of alleles at only three genes RSPO2, FGF5, and KRT71 and thus possesses long, curly hair with furnishings  pathway. Variants result from mutations in the microphthalmia-associated transcription factor (MITF), which is crucial for melanocyte migration Several dog breeds exhibit complete or partial absence of pigmentation. For instance, Karlsson et al. mapped a locus for white-spotting to a 102-kb haplotype on CFA 20 in a region that spans a single gene; The identification of genetic variants controlling morphology in the dog population has reached an exciting juncture. The current set of available molecular tools allows us to finally address the critical questions. For instance, the striking morphological variation observed between breeds of dogs provides us with unique opportunities to study the genetic basis of both evolution and domestication. A deeper understanding of the genomics and variation in wild canids would enhance our ability to pursue these questions.Several hypotheses have been proposed as to why the dog, as opposed to any other domestic land mammal or any other domesticated creature, displays such extremes of morphologic variation (Box 1). Each theory has its champions, and most likely a combination of mechanisms contribute with the strong artificial selection imposed by man being the most important. The question remains: if under the same intense artificial selection for novel morphological traits, would other domestic creatures exhibit equivalent variation? As scientists continue hunting for the genes and tracking the mutations that control morphologic variation in the domestic dog, we expect still more secrets will be revealed regarding the genetic basis of man's extraordinary best friend.Microsatellites or simple sequence repeatsHigh levels of repeat purityThe abundance and location of SINEC_Cf elements in the canine genomeCommon sources of variation: mutational hotspots, chromosomal fissions, and gene duplicationsIntense artificial selectionRapid perpetuation of new mutations


Heritability in mate preferences is assumed by models of sexual selection, and preference evolution may contribute to adaptation to changing environments. However, mate preference is difficult to measure in natural populations as detailed data on mate availability and mate sampling are usually missing. Often the only available information is the ornamentation of the actual mate. The single long-term quantitative genetic study of a wild population found low heritability in female mate ornamentation in Swedish collared flycatchers. One potentially important cause of low heritability in mate ornamentation at the population level is reduced mate preference expression among inexperienced individuals.Applying animal model analyses to 21 years of data from a Hungarian collared flycatcher population, we found that additive genetic variance was 50 percent and significant for ornament expression in males, but less than 5 percent and non-significant for mate ornamentation treated as a female trait. Female breeding experience predicted breeding date and clutch size, but mate ornamentation and its variance components were unrelated to experience. Although we detected significant area and year effects on mate ornamentation, more than 85 percent of variance in this trait remained unexplained. Moreover, the effects of area and year on mate ornamentation were also highly positively correlated between inexperienced and experienced females, thereby acting to remove difference between the two groups.The low heritability of mate ornamentation was apparently not explained by the presence of inexperienced individuals. Our results further indicate that the expression of mate ornamentation is dominated by temporal and spatial constraints and unmeasured background factors. Future studies should reduce unexplained variance or use alternative measures of mate preference. The heritability of mate preference in the wild remains a principal but unresolved question in evolutionary ecology. High genetic variability is common in sexually selected traits However, directional mate choice also shows variation within a single population. For example, females that are unattractive or in poor condition may need to be less choosy due to the risk of rejection by males Theoretical models that laid the foundation of contemporary sexual selection research examined the evolution of mating preferences, which means that they assumed heritable variation in preferences  fathers and sons were found to positively correlate in the ornamentation of females they mated with, and mate ornamentation was also repeatable within males. In this study, additive genetic and other individual-specific (i.e. permanent environment) effects on mate attractiveness could not be distinguished. The second study was conducted in a Swedish population of collared flycatchers  on the island of Gotland, using an extensive, long-term pedigree To the best of our knowledge, there are only two studies of the inheritance of mating patterns in the wild. Both studies used the ornamentation of the mate as a measure of an individual's mate preference. In the first study Studies in many species have suggested that mate choice is age-dependent Here we concentrate on the effect of breeding experience on the heritability of mate ornamentation. As a first step, we look for effects of breeding experience on breeding date and clutch size when controlling for female age, to see whether our coding of experience is meaningful. As a second step, we repeat the population-level heritability analyses conducted in the Swedish population to see whether the apparent additive genetic background of mate FPS is similar in our population. For comparison, we also estimate the variance components of FPS in males  We ignore other sexual traits such as wing patch size and song throughout, due to the lack of adequate data from those traits for this analysis. However, the independent treatment of FPS is justified by the fact that this ornament, with its low phenotypic plasticity, occupies a special, disjunct position among male sexual traits in our population 1,4078 = 86.57, p<0.001), but less strongly affected by age . Inexperienced females laid eggs much later than experienced ones  and age . The same was true for date-corrected clutch size .Breeding date (n = 5651 observations) was strongly influenced by experience (Fced ones . Clutch A), while variances due to year (VYEAR) and nestbox plot (VPLOT) were also significant and together explained another ten percent of variation. VPE was not significant. When analyzing female mate FPS , but it had a broad error range and therefore did not differ significantly from zero either . In this bivariate model, VA seemed slightly higher in the inexperienced group, but it was not significantly different from zero in either category . Simultaneously constraining rG as 1.0 and VA in the two experience categories as equal did not lead to a significantly different model likelihood compared to the unconstrained model . In sum, there was no evidence that experience affected the additive genetic background of mate FPS. However, the correlation of nestbox plot effects on mate FPS between experience categories (rPLOT) was bound to 1.0 while the correlation of year effects (rYEAR) was 0.921±0.098. Both rPLOT and rYEAR were significantly different from zero .Our final analysis was a bivariate animal model of mate FPS among inexperienced and experienced females. Similar to the univariate results, VA) for ornamentation (FPS) as a male trait (fifty percent). For mate FPS as a female trait, however, VA was very low (less than five percent), and breeding experience had no demonstrable effect of on the expression of additive genetic variation. We also found significant spatial and temporal constraints on both the expression of mate FPS and its pattern with experience, and a large percentage of unexplained variance in mate FPS. These results raise questions about the adequate quantification of mate preferences in wild populations.We detected ample additive genetic variation . Therefore, if an inexperienced and an experienced female shared breeding area or year, this made their mate FPS similar. At the population level, the numbers of available data varied vastly among combinations of year and nestbox plot, so the high rPLOT and rYEAR we found also imply that temporal and spatial heterogeneity tended to blur any existing experience effect on mate FPS. In other words, temporal and spatial constraints not only affect the distribution of mate FPS, but may also limit the detectability of other, functionally independent, biologically meaningful effects on this trait.These limiting factors include temporal changes in the ornamentation of available males Therefore, mate ornamentation seems to be a poor measure of mate preferences in our population, and this makes its heritability and patterns with experience difficult to interpret. In response to a similar critique To summarize, it seems that the limiting factor in the correlative approach introduced by Ref. All work was conducted with ringing license from the Hungarian Ornithological and Nature Conservation Society , long-term research agreements with the Pilis Park Forestry (December 1988 and March 2007) and research permits from Duna-Ipoly National Park and the regional nature conservation authority .The fieldwork was conducted in the Pilis Mountains, near Szentendre, Hungary, where a nestbox breeding population  of collared flycatchers has been intensively monitored since 1982. More details on the population and the study site have been presented elsewhere In our population, extrapair paternity does not seem to be consistently related to any male trait, including FPS The comprehensive 21-year phenotypic dataset from which we drew our data contained n = 4233 male FPS records and n = 3726 female mate FPS records. Repeated records of individuals within years and all broods where brood size had been manipulated or the nestlings had been cross-fostered without individual identification were deleted from the analyses. Of the remaining data, those that could be used for the pedigree analyses (recruits and their parents) consisted of n = 2138 male FPS records and n = 1971 female mate FPS records from n = 1380 recruits and their parents .F statistics. Importantly, estimates of additive genetic variance and covariance in an animal model depend on the other random and fixed effects in the model Data were analyzed using ASReml2  to fit a series of animal models. An animal model is a linear mixed effect model that includes individual genetic merit as a random effect such that, in the presence of pedigree data, phenotypic variance can be partitioned into (additive) genetic and environmental components Firstly, we looked for effects of female breeding experience on breeding date (log-transformed first egg date relative to yearly median) and clutch size using general linear mixed models in PROC MIXED of SAS 9.1 (SAS Institute). The effect of breeding experience is confounded by age, so we tested experience effects in two ways: with binary age as a simultaneous predictor, and in the subset of after-second-year (i.e. old) females. The two approaches always gave the same results, so we report results of the former, which relies on all available data and is therefore more powerful. All models contained female identity, nestbox plot (i.e. breeding area) and year as random effects, and female age and experience as fixed factors. For clutch size, we also ran a model with breeding date as a covariate to see whether experience affects primary reproductive output when controlling for differences in breeding date.A), permanent environment (VPE), breeding area (VPLOT), year of breeding (VYEAR), and residual (VR) components. Plot and year effects were included to account for expected spatial and temporal heterogeneity in the environment. The permanent environment effect makes use of repeated measures available from individuals to account for fixed non-genetic differences between individuals that can otherwise bias the estimation of VA. Phenotypic variance VP was determined as the sum of the variance components and heritability estimated as the ratio of VA to VP. Note that this model is similar to that used by Ref. Secondly, we estimated variance components for female mate FPS and male FPS. For female mate FPS, the model included binary mate age  and binary breeding experience  as fixed factors. For male FPS, the only fixed factor was the binary age of the male. Male age as a fixed effect was included because FPS is weakly but significantly age-dependent G. The area- and year-generated covariances between the experience categories were modeled as well . As an explicit test for a genotype by-breeding experience interaction we compared the likelihood of this model to one in which rG was set to unity and VA was constrained to be constant across experience classes.Thirdly, to test the null hypothesis that the heritability of mate ornamentation does not change with female experience, we ran a bivariate animal model in which the mate FPS of inexperienced and experienced females were modeled as two separate traits. We used the above model structure except that the permanent environment component was fit only in the experienced group. This is because inexperienced birds were defined as those in their first breeding attempt and therefore there can be no repeated measures of this trait. Heritabilities for each trait were estimated as well as the genetic covariance between them which was rescaled to estimate the genetic correlation r